Disruptions of the myofiber cytoskeleton underlie several genetic muscular dystrophies, including Duchenne and Limb-girdle muscular dystrophies. In addition to these dystrophin-related disorders, certain inherited muscular dystrophies are due to mutations in cytoskeletal proteins that do not interact with the dystrophin complex. Identification of the responsible proteins and clarification of mechanisms that regulate the myofiber cytoskeleton are therefore critical goals. Recent molecular cloning studies have identified actinin-associated LIM protein (ALP), which is a novel component of the muscle cytoskeleton. ALP contains a PDZ protein motif that is also present in certain dystrophin-associated proteins, yet ALP does not interact with dystrophin. Instead ALP binds to actinin, a structural homologue of dystrophin, and ALP associates with actinin at the Z-lines of skeletal muscle. Chromosomal mapping studies show that ALP occurs in 4q35, within 7 Mb of the telomeric region that is deleted in facioscapulohumeral muscular dystrophy (FSHD), the most common autosomal muscular dystrophy. ALP is the only muscle-specific gene yet found to map in this region. Therefore, a possible role for ALP in the pathogenesis of FSHD must be explored. We now propose to characterize the molecular interaction of ALP with actinin and to determine the composition and function of the ALP-associated complex at the Z-lines. To help assess whether ALP participates in FSHD, we will determine whether ALP expression is altered in muscle biopsies from diseased patients. Because FSHD is a dominant disease, it is likely that only one allele of the responsible gene(s) will be abnormal. To address this, we will also evaluate allele-specific expression of ALP in FSHD muscle samples. Because complex genetics underlie FSHD, studies of human tissues alone may not decisively identify the responsible gene(s). We will therefore target disruption of ALP in stem cells and breed mice that lack ALP protein. Muscle development, histology and function will be carefully evaluated in the ALP mutants. Assembly of the ALP-associated protein complex at the Z-lines will also be evaluated in the mutants. If these mutant mice manifest signs that resemble FSHD this would implicate a role for ALP in this disease and the mice would provide a unique animal model. The proposed studies will lead to a better understanding of formation and function of the myofiber cytoskeleton and may provide insight in the pathogenesis and treatment of FSHD.